Constitutive and barbital-induced expression of the Cyp6a2 allele of a high producer strain of CYP6A2 in the genetic background of a low producer strain

The levels of one or more cytochrome P450 (CYP) enzymes and the respective mRNAs are found to be higher in insecticide-resistant insects than in susceptible insects. To understand better how insects regulate the levels of CYPs, we examined the expression of the Cyp6a2 gene in various strains of Drosophila melanogaster. We also took a transgenic approach to understand the molecular mechanisms that are involved in strain variation of Cyp6a2 expression. RNA blot analysis showed that the constitutive expression of Cyp6a2 varies from strain to strain; the level of CYP6A2 mRNA is barely detectable in the underproducer ry⁵⁰⁶ strain, whereas it is very high in the overproducer 91-R and MHIII-D23 strains. The long terminal repeat (LTR) of mobile element 17.6 that is found in the 3′ untranslated region (UTR) of the Cyp6a2 gene of some strains does not appear to have any role on the steady-state CYP6A2 mRNA level. We also found that the Cyp6a2 gene is inducible by barbital in 91-R, ry⁵⁰⁶ as well as 91-C, which carries an LTR insertion. To examine the genetic background of the underproducer ry⁵⁰⁶ strain with respect to Cyp6a2 expression, we transformed the ry⁵⁰⁶ strain with the Cyp6a2 allele of the overproducer 91-R strain (Cyp6a2-91R) and measured the constitutive and barbital-induced expression of the Cyp6a2-91R transgene in the transformed flies. The Cyp6a2-91R transgene carrying 129 bp of DNA upstream of the ATG codon did not show any constitutive or barbital-induced expression in the ry⁵⁰⁶ host genome. However, transgenes with 1331 and 985 bp upstream DNA showed similar levels of constitutive expression that were higher than that of the endogenous Cyp6a2 gene of the ry⁵⁰⁶ host strain, but lower than the expression of the same gene in the 91-R strain. Both these transgenes, with 1331 and 985 bp upstream DNA, also showed induction with 0.1 M barbital. DNA sequence analysis revealed that in both 91-R and ry⁵⁰⁶, the upstream DNA between +1 and −985 bp contains a distal and a proximal group of three potential barbie boxes, i.e. cis-elements that are thought to be involved in barbiturate-mediated induction of CYP genes. Except for four bases located near the distal cluster of barbie boxes and two other bases, the base sequence of the upstream DNA is identical in ry⁵⁰⁶ and 91-R strains. These results suggest that the underproducer ry⁵⁰⁶ strain has the trans-regulatory factors to support constitutive and induced expression of the Cyp6a2-91R allele carrying DNA between −129 and −1331 bp regions. Possible reasons for low constitutive expression of the endogenous Cyp6a2 gene and moderate level of expression of the Cyp6a2-91R allele in the ry⁵⁰⁶ genetic background are discussed.     

An antibiotic selection marker for schistosome transgenesis

Drug selection is widely used in transgene studies of microbial pathogens, mammalian cell and plant cell lines. Drug selection of transgenic schistosomes would be desirable to provide a means to enrich for populations of transgenic worms. We adapted murine leukaemia retrovirus vectors – widely used in human gene therapy research – to transduce schistosomes, leading to integration of transgenes into the genome of the blood fluke. A dose–response kill curve and lethal G418 (geneticin) concentrations were established: 125–1,000 μg/ml G418 were progressively more toxic for schistosomules of Schistosoma mansoni with toxicity increasing with antibiotic concentration and with duration of exposure. By day 6 of exposure to ?500 μg/ml, significantly fewer worms survived compared with non-exposed controls and by day 8, significantly fewer worms survived than controls at ?250 μg/ml G418. When schistosomules were transduced with murine leukaemia retrovirus encoding the neomycin resistance (neoR) transgene and cultured in media containing G418, the neoR transgene rescued transgenic schistosomules from the antibiotic; by day 4 in 1,000 μg/ml and by day 8 in 500 μg/ml G418, significantly more transgenic worms survived the toxic effects of the antibiotic. More copies of neoR were detected per nanogram of genomic DNA from populations of transgenic schistosomes cultured in G418 than from transgenic schistosomes cultured without G418. This trend was G418 dose-dependent, demonstrating enrichment of transgenic worms from among the schistosomules exposed to virions. Furthermore, higher expression of neoR was detected in transgenic schistosomes cultured in the presence of G418 than in transgenic worms cultured without antibiotic. The availability of antibiotic selection can be expected to enhance progress with functional genomics research on the helminth parasites responsible for major neglected tropical diseases.     

Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.     

Structural attributes of nucleotide sequences in promoter regions of supercoiling-sensitive genes: How to relate microarray expression data with genomic sequences

The level of supercoiling in the chromosome can affect gene expression. To clarify the basis of supercoiling sensitivity, we analyzed the structural features of nucleotide sequences in the vicinity of promoters for the genes with expression enhanced and decreased in response to loss of chromosomal supercoiling in Escherichia coli. Fourier analysis of promoter sequences for supercoiling-sensitive genes reveals the tendency in selection of sequences with helical periodicities close to 10 nt for relaxation-induced genes and to 11 nt for relaxation-repressed genes. The helical periodicities in the subsets of promoters recognized by RNA polymerase with different sigma factors were also studied. A special procedure was developed for the study of correlations between the intensities of periodicities in promoter sequences and the expression levels of corresponding genes. Significant correlations of expression with the AT content and with AT periodicities about 10, 11, and 50 nt indicate their role in regulation of supercoiling-sensitive genes.     

In vivo evidence that Agxt2 can regulate plasma levels of dimethylarginines in mice

Elevated plasma concentrations of the asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse cardiovascular clinical outcomes. Both dimethylarginines can be degraded by alanine–glyoxylate aminotransferase 2 (Agxt2), which is also the key enzyme responsible for the degradation of endogenously formed β-aminoisobutyrate (BAIB). In the present study we wanted to investigate the effect of BAIB on Agxt2 expression and Agxt2-mediated metabolism of dimethylarginines. We infused BAIB or saline intraperitoneally for 7 days in C57/BL6 mice via minipumps. Expression of Agxt2 was determined in liver and kidney. The concentrations of BAIB, dimethylarginines and the Agxt2-specific ADMA metabolite α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) was determined by LC–MS/MS in plasma and urine. As compared to controls systemic administration of BAIB increased plasma and urine BAIB levels by a factor of 26.5 (p < 0.001) and 25.8 (p < 0.01), respectively. BAIB infusion resulted in an increase of the plasma ADMA and SDMA concentrations of 27% and 31%, respectively, (both p < 0.05) and a 24% decrease of plasma DMGV levels (p < 0.05), while expression of Agxt2 was not different.Our data demonstrate that BAIB can inhibit Agxt2-mediated metabolism of dimethylarginines and show for the first time that endogenous Agxt2 is involved in the regulation of systemic ADMA, SDMA and DMGV levels. The effect of BAIB excess on endogenous dimethylarginine levels may have direct clinical implications for humans with the relatively common genetic trait of hyper-β-aminoisobutyric aciduria.     

Serum androgen level is determined by autosomal dominant inheritance and regulates sex-related CYP genes in pigs

We have previously demonstrated differences between Meishan and Landrace pigs in their serum androgen levels (Meishan > Landrace) and the expression of genes encoding hepatic cytochrome P450 (CYP) 1A subfamily enzymes (Meishan < Landrace). In the present study, to clarify whether such differences are genetically controlled, we crossbred these pigs (female Meishan × male Landrace, ML; female Landrace × male Meishan, LM) and examined the expression levels of serum androgen and hepatic CYP family genes (CYP1A1, CYP1A2, CYP2A19, and CYP2E1) among ML, LM, and their parents. In sexually mature (5-month-old) male ML or LM pigs, not only the serum androgen level, but also the hepatic expression levels of all the CYPs examined were similar to those in male Meishan pigs. In addition, there were few breed differences among the females of Meishan, Landrace, ML and LM pigs in the expression of all the CYP genes examined. Furthermore, the expression levels of these CYPs in the females of Meishan and Landrace pigs could be decreased to the corresponding levels in male Meishan pigs by administration of testosterone propionate. The present findings demonstrate that serum androgen level is determined by autosomal dominant inheritance and that the level of serum androgen is one of the host factors regulating the constitutive expression of CYP1A1, CYP1A2, CYP2A19, and CYP2E1 in the pig liver.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

High titer production of tetracenomycins by heterologous expression of the pathway in a Streptomyces cinnamonensis industrial monensin producer strain

Streptomyces cinnamonensis C730.1 and C730.7, are industrially mutagenized strains that produce moderate and high levels of the polyketide polyether antibiotic monensin A, respectively, in an oil-based fermentation medium. The possibility that these strains could be used for high titer production of a heterologous polyketide product was investigated by expression of the entire tetracenomycin (TCM) biosynthetic pathway using an integrative plasmid, pSET154. Expression in C730.1 led to stable production of ~0.44 g/l TCM C (the final biosynthetic product) and ~2.69 g/l TCM A2 (the penultimate biosynthetic product), and resulted in a 40% decrease in monensin production. Expression in the C730.7 led to higher levels of TCMs, ~0.6 g/l TCM C and ~4.35 g/l TCM A2, without any detectable decrease in the higher titer monensin production. Abrogation of monensin production in this strain through deletion of the corresponding biosynthetic genes did not lead to higher levels of TCM products. In the case of the C730.7 host, 85% of the TCM C and virtually all of the TCM A2 were intracellular, suggesting feedback inhibition leads to the accumulation of the final pathway intermediate. These observations contrast those made for the native producer Streptomyces glaucescens where the predominant product is TCM C and TCM titers are significantly lower levels (~0.3 g/l), and demonstrate the potential utility of S. cinnamonensis strains as heterologous hosts for high level expression of a variety of polyketide synthase derived products.     

Oral cancer,cigarette smoke and mitochondrial 18 kDa translocator protein (TSPO) — In vitro,in vivo,salivary analysis

Oral cancer features high rates of mortality and morbidity, and is in dire need for new approaches. In the present study we analyzed 18 kDa translocator protein (TSPO) expression in oral (tongue) cancer tumors by immunohistochemistry. We also assayed TSPO binding in human tongue cancer cell lines and in the cellular fraction of saliva from tongue cancer patients, heavy cigarette smokers, and non-smoking healthy people as controls. Concurrently, TSPO protein levels, cell viability, mitochondrial membrane potential (Δψ_m), and general protein levels were analyzed. TSPO expression could be significantly enhanced in oral cancer tumors, compared to unaffected adjacent tissue. We also found that five-year survival probability dropped from 65% in patients with TSPO negative tumors to 7% in patients with highly expressed TSPO (p < 0.001). TSPO binding capacity was also pronounced in the human oral cancer cell lines SCC-25 and SCC-15 (3133 ± 643 fmol/mg protein and 6956 ± 549 fmol/mg protein, respectively). Binding decreased by 56% and 72%, in the SCC-25 and SCC-15 cell lines, respectively (p < 0.05) following CS exposure in cell culture. In the cellular fraction of saliva of heavy smokers TSPO binding was lower than in non-smokers (by 53%, p < 0.05). Also the cellular fraction of saliva exposed to CS in vitro showed decreased TSPO binding compared to unexposed saliva (by 30%, p < 0.001). Interestingly, oral cancer patients also displayed significantly lower TSPO binding in the cellular fraction of saliva compared to healthy controls (by 40%, p < 0.01). Our results suggest that low TSPO binding found in the cellular fraction of saliva may depend on genetic background as well as result from exposure to CS. We suggest that this may be related to a predisposition for occurrence of oral cancer.     

Gene expression of apoptosis-related genes,stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.     

The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index

Ghrelin is the only known peripherally produced and centrally acting peptide hormone stimulating food intake. The acylation of ghrelin is essential for binding to its receptor. Recently, the ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) was identified in mice, rats and humans. In addition to gastric mucosal expression, GOAT was also detected in the circulation of rodents and its expression was dependent on metabolic status. We investigated whether GOAT is also present in human plasma and whether expression levels are affected under different conditions of body weight. Normal weight, anorexic and obese subjects with body mass index (BMI) 30–40, 40–50 and >50 were recruited (n = 9/group). In overnight fasted subjects GOAT protein expression was assessed by Western blot and ghrelin measured by ELISA. GOAT protein was detectable in human plasma. Anorexic patients showed reduced GOAT protein levels (−42%, p < 0.01) whereas obese patients with BMI > 50 had increased concentrations (+34%) compared to normal weight controls. Ghrelin levels were higher in anorexic patients compared to all other groups (+62–78%, p < 0.001). Plasma GOAT protein expression showed a positive correlation with BMI (r = 0.71, p < 0.001) and a negative correlation with ghrelin (r = −0.60, p < 0.001). Summarized, GOAT is also present in human plasma and GOAT protein levels depend on the metabolic environment with decreased levels in anorexic and increased levels in morbidly obese patients. These data may indicate that GOAT counteracts the adaptive changes of ghrelin observed under these conditions and ultimately contributes to the development or maintenance of anorexia and obesity as it is the only enzyme acylating ghrelin.     

Genetic diversity and population structure in Vallisneria spinulosa (Hydrocharitaceae)

Vallisneria spinulosa is a dominant submerged macrophyte in lakes of the middle–lower reaches of the Yangtze River. Allozyme variation, clonal diversity and population genetic structure were investigated for a total of 396 individuals sampled from 10 extant populations. V. spinulosa maintained high levels of genetic variation both at the species (P = 46.2, A = 1.69, H_e = 0.23) and at the population level (P = 46.2, A = 1.58, H_e = 0.21). Although aquatic macrophytes commonly exhibit low genetic variation within populations, the obligately outcrossing mating system of V. spinulosa and pervasive gene flow likely account for the high levels of diversity maintained within populations. All V. spinulosa populations contained high clonal diversity with a mean proportion of distinguishable genotypes of 0.57 and a mean Simpson's diversity index of 0.95, indicating that populations were founded sexually or that successful seedling recruitment occurred after initial colonization. Partitioning of genetic diversity revealed a surprisingly low population differentiation (G_ST = 0.06) as compared to other hydrophilous angiosperms. No evidence of isolation-by-distance was found (r = 0.056, P = 0.312), suggesting that gene flow was not restricted geographically. The UPGMA cluster analysis revealed that several widely separated populations grouped together, suggesting long-distance gene flow among populations. The high vagility of V. spinulosa and extensive hydrologic connectivity among populations have facilitated long-distance gene flow and resulted in the pattern of population genetic structure in V. spinulosa.     

Population structure and genetic diversity of Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an endangered and endemic species in China. To provide scientific foundation for formulating conservation strategies, we sampled six extant populations of this species and assessed the levels and patterns of genetic diversity using ISSR markers (11 primers). Of 144 bands detected 57.64% were polymorphic, but on average only 20.72% were polymorphic within populations. Our results revealed a low level of intraspecific genetic diversity (at population level: H_pop = 0.082, H_B = 0.177, SI = 0.1194; at species level: H_pop = 0.207, H_B = 0.378, SI = 0.3069). A high level of genetic differentiations among populations was detected based on Nei's genetic diversity analysis (60%), AMOVA analysis (65%), and Bayesian analysis (53%). The low levels of heterozygosity and high genetic differentiation observed in D. versipellis may be the consequence of low rate of natural recruitment, clonal growth, gene drift, and habitat fragmentation. Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species. Ex situ conservation should sample from different populations across the distribution range of the species to conserve high genetic diversity.     

The effects of phytase transgenic maize on the community components and diversity of arthropods

It is important to understand the effect of phytase transgenic (PT) maize on arthropod communities in natural ecosystems. In this study, a 2-year survey of arthropod community biodiversity in fields of PT maize (0 7 8) and non-genetically modified (RA119, non-GM) maize was performed using sweep-net sampling on the stems and leaves of the maize plants. The results showed that there was no significant difference in the individual number of herbivorous, predatory, neutral and parasitic groups in PT maize and non-GM maize. The species number of herbivorous group in PT maize was significantly lower than that in non-GM maize in 2013 (p < 0.05). The proportions of different arthropod groups were almost identical in the PT maize and non-GM maize in terms of both species and individual number. Moreover, there was no significant difference in the Shannon-Weiner diversity index (H'), evenness index (J), dominance index (D), richness (S), and species abundance (N) between the two types of maize. The similarity coefficient of the arthropod community suggested that the arthropod community composition of PT maize was similar to that of non-GM maize. Furthermore, PT maize had no significant effect on the relative stability of the arthropod community. These results indicated that despite the presence of a relatively minor difference in arthropod community between the PT maize and non-GM maize, the PT maize had little effect on arthropod community biodiversity.     

Potential of Lariophagus distinguendus (Förster) (Hymenoptera: Pteromalidae) to suppress the maize weevil Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in bagged and bulk stored maize

Grains are often stored in jute bags in developing countries, especially in Africa, as well as in small quantities in bulk. Parasitoids suitable for biological control of stored-product pests should be able to find their hosts in bulk grain or in jute bags over a certain distance in a warehouse containing stacks of bagged grain. The potential of using Lariophagus distinguendus for the biological control of Sitophilus zeamais was assessed in maize stored in jute bags and bulk grain. The ability of the parasitoid to penetrate the jute cloth and the grain mass and parasitize its host was studied under controlled conditions of 25 ± 1 °C and 65 ± 5% RH. Experiments were carried out in small 5-kg jute bags containing 28 d old S. zeamais larvae within infested maize kernels, and in cylinders filled with maize grains and containing caged hosts at different depths. L. distinguendus parasitized S. zeamais in the jute bags and in the storage cylinders at various depths. In the jute bag experiment, out of the 60 L. distinguendus adults released, a mean ± SD of 7.03 ± 1.78% and 6.34 ± 1.01% of the 40 females and of the 20 males released, respectively, entered the jute bags. Significantly, no differences were found between the female and male L. distinguendus that entered the bags. Mean reduction of S. zeamais in the jute bags by parasitoids was 81%. The parasitic wasps also significantly reduced the emergence of S. zeamais in bulk maize. At depths of 20–45 cm from the grain surface, mean reduction of S. zeamais was 74%, while from 95 to 100 cm, mean reduction was 34%. When results from depths lower than 50 cm were pooled and compared with pooled data from depths higher than 90 cm, there was a significant reduction in parasitism at depths of more than 90 cm. For depths below 50 cm a mean of 5.3 L. distinguendus adult offspring per cage emerged compared with a mean of 2.6 at depths of more than 90 cm. These results support the approach to utilize L. distinguendus as a component in the integrated control of S. zeamais in bagged or bulk stored maize.